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Abstract:

The present invention provides novel chimeric receptors and methods of
screening using the chimeric receptors. The chimeric receptors comprise
an extracellular domain of a tumor necrosis factor receptor superfamily
(TNFRSF) receptor and an intracellular domain with kinase activity
stemming from a receptor tyrosine kinase. According to an embodiment, the
chimeric receptor comprises a full-length TNFRSF receptor. The present
invention provides means for screening and testing of modulators of
TNFRSF receptors.

Claims:

1. A chimeric polypeptide comprising: a first part comprising an amino
acid sequence that is substantially identical to the amino acid sequence
of an extracellular, ligand-binding portion of a receptor A, said
receptor A being selected from receptors of the tumor necrosis factor
receptor super family (TNFRSF); a second part comprising an amino acid
sequence that is substantially identical to the amino acid sequence of an
intracellular, signalling kinase portion of a receptor B, said receptor B
being selected from receptor tyrosine kinases (RTKs); and, between said
first and second parts, a third part comprising an amino acid sequence
taken from and/or substantially identical to a transmembrane domain.

2. The chimeric polypeptide of claim 1, wherein the amino acid sequence
of said first part has the capacity of oligomerization with the
corresponding extracellular domain of the receptor A and/or with another
chimeric polypeptide of claim 1.

3. The chimeric polypeptide of claim 1, wherein the amino acid sequence
of said first part has the capacity of binding an agent exhibiting an
activity on receptor A, such as a natural ligand of the receptor A.

4. The chimeric polypeptide of claim 1, wherein the amino acid sequence
of said second part has the capacity of oligomerization with the
corresponding intracellular domain of the receptor B and/or of another
chimeric polypeptide of claim 1.

5. The chimeric polypeptide of claim 1, wherein the amino acid sequence
of said second part has tyrosine kinase activity following dimerization.

6. The chimeric polypeptide of claim 1, wherein said transmembrane domain
is selected from transmembrane domains of receptors of the TNFRSF and of
RTKs.

10. The chimeric polypeptide of claim 1, which comprises an amino acid
sequence taken from or substantially identical to a death domain.

11. The chimeric polypeptide of claim 10, wherein said death domain has
an amino acid sequence that is substantially identical to the death
domain of TNFRSF1.

12. The chimeric polypeptide according to claim 1, which comprises an
extracellular, ligand-binding portion of a TNFRSF receptor, a
transmembrane domain, and an intracellular, signalling kinase portion of
an RTK.

14. A cell expressing the nucleotide sequence as defined in claim 13,
and/or in the plasma membrane of which is embedded the encoded chimeric
polypeptide.

15. A method of screening agents which are capable of affecting the
activity of a receptor A selected from receptors of the tumor necrosis
factor receptor super family (TNFRSF), said method comprising the steps
of: providing cells expressing at least one nucleotide sequence encoding
a chimeric polypeptide comprising: a first part comprising an amino acid
sequence that is substantially identical to the amino acid sequence of an
extracellular, ligand-binding portion of a receptor A, said receptor A
being selected from receptors of the tumor necrosis factor receptor super
family (TNFRSF); a second part comprising an amino acid sequence that is
substantially identical to the amino acid sequence of an intracellular,
signalling kinase portion of a receptor B, said receptor B being selected
from receptor tyrosine kinases (RTKs); and, between said first and second
parts, a third part comprising an amino acid sequence taken from and/or
substantially identical to a transmembrane domain; exposing a candidate
agent to be screened to said cells; measuring a physical, biological
and/or chemical value that is associated with a cellular condition of
said cells; and determining, from the value measured in the preceding
step, if said candidate agent is an agent that is capable of affecting
the activity of said receptor A.

16. The method of claim 15, wherein an agent affects the activity of a
receptor if it affects a status of signalling of the receptor.

17. The method of claim 15, wherein said candidate is an active agent of
said receptor A, if it affects said cellular condition of said cells.

18. The method of claim 15, wherein said cellular condition is at least
partly dependent of an activity and/or a condition of said chimeric
polypeptide.

19. The method of claim 15, wherein said cellular condition is at least
partly dependent of activity or absence of activity of the intracellular
kinase domain of said chimeric polypeptide.

20. The method of claim 15, wherein said cellular condition is
concentration or a change in the concentration of one or more selected
from the group consisting of: intracellular Ca2+, inositol phosphate
(IP1) and inositol triphosphate (IP3).

21. The method of claim 15, wherein said physical, biological and/or
chemical value that is associated with a cellular characteristic is
fluorescence or luminescence or both.

22. (canceled)

23. A chimeric polypeptide comprising: an amino acid sequence that is
substantially identical to the amino acid sequence of the extracellular,
ligand binding portion of a receptor A, said receptor A being selected
from receptors of the TNFRSF, a transmembrane domain; optionally, an
amino acid sequence that is substantially identical to the amino acid
sequence of a death domain; and, an amino acid sequence that is
substantially identical to the amino acid sequence of an intracellular,
signalling kinase portion of a receptor B, said receptor B being selected
from receptor tyrosine kinases (RTKs).

Description:

THE FIELD OF THE INVENTION

[0001] The present invention relates to the field of drug discovery and
drug screening and to the development of assays useful in drug screening.
More specifically, the present invention relates to methods of screening
agents that are capable of affecting the activity of receptors of the
tumor necrosis factor receptor superfamily. The present invention further
relates to polypeptides, nucleic acids, vectors and cells, which may be
used in such methods.

BACKGROUND OF THE INVENTION AND PROBLEMS TO BE SOLVED BY THE INVENTION

[0002] As currently practiced in the art, drug discovery is a long and
multiple step process involving identification of specific disease
targets, development of an assay based on a specific target, validation
of the assay, optimization and automation of the assay to produce a
screen, high-throughput screening (HTS) of compound libraries using the
assay to identify "hits", hit validation and hit compound optimization.
The output of this process is a lead compound that goes into pre-clinical
and, if validated, eventually into clinical trials. In this process, the
screening phase is distinct from the assay development phases, and
involves testing compound efficacy in living biological systems.

[0003] The conventional measurement in early drug discovery assays used to
be radioactivity. However, the need for more information, higher
throughput and miniaturization has caused a shift towards using
fluorescence and/or luminescence detection. Fluorescence-based reagents
can yield more powerful, multiple parameter assays that are higher in
throughput and information content and require lower volumes of reagents
and test compounds. Fluorescence is also safer and less expensive than
radioactivity-based methods. Automatized fluorescence plate readers
(FLIPR) have been extensively used in the context of drug discovery to
measure fluorescence in the context of HTS. In particular,
fluorescence-based, quantitative reliable and time-resolved HTS methods
have been developed for chemical active agents of G-protein coupled
receptors (GPCRs).

[0004] However, for tumor necrosis factor receptor superfamily (TNFRSF),
dynamic and quantitative drug screen systems have not yet been
established. Signalling TNFRSF members are characterized by an
extracellular N terminal region including one to six cysteine-rich
domains (CRDs), an intracellular C terminus and a single hydrophobic
transmembrane spanning domain. The problems of providing efficient
screening systems with these types of receptors may be associated with
the particular mechanisms and interactions involved in ligand binding and
signal transduction, generally requiring oligomerization of the receptor
and often involving oligomerized ligands.

[0009] Thus, in the absence of ligand, a number of TNFRSF members exist in
the form of homodimers. Upon ligand engagement, each pre-assembled dimer
has the capacity to engage two trimeric ligands and therefore may form
molecular bridges between trimers leading to receptor trimers
aggregation.

[0010] Tumor necrosis factor (TNF), the natural ligand of tumor necrosis
factor receptor 1 and 2 (TNFR1 and TNFR2/TNFRSF1B, respectively), plays a
central in the pathogenesis of inflammatory diseases and neutralizing
monoclonal antibodies against TNF (such as Infliximab and Adalimumab) or
soluble TNFR-immunoglobulin fusion proteins (such as Etanercept) have
been successfully used in the treatment of diseases such as rheumatoid
arthritis, ankylosing spondylitis, psoriasis, and psoriatic arthritis.

[0011] As detailed further below, abnormal levels of TNFSFs have been
shown to be implicated in many disorders and disease conditions,and thus
there is an interest in developing an assay allowing for quantitative and
dynamic HTS of agents exerting an activity on receptors of this family.

[0012] As of today, proteins constitute the only therapeutic modality for
targeting TNFRSFs. Protein therapeutics have drawbacks such as route of
administration (they are injectables), high cost of production, and
development of antibodies, among others (Semin Cutan Med Surg. March
2007; 26(1):6-14). There is therefore a need to identify alternate and
improved drugs, such as small molecule inhibitors, for the treatment of
disorders involving TNFSFs. Small molecules present the advantage of
being orally available with convenience of use and increased patient
compliance, non-immunogenicity, and lower manufacturing costs. Small
molecules have also the potential to cross the blood-brain barrier and
treat pathologies of the central nervous system (CNS) otherwise not
accessible to large proteins such as antibodies and recombinant
receptors.

[0020] Monitoring production of TNF-induced NF-κB
target genes such as Interleukin-1, Interleukin-6 or Interleukin-8 by
ELISA;

[0021] Monitoring cytotoxicity and cell death by activation of
caspases.

[0022] However, these methods have several drawbacks: They measure events
distal to the target receptor, and/or they are cumbersome and not
amenable to HTS, and/or they do not measure target-specific events. In
general, these prior art methods are not suitable for rapid, dynamic and
quantitative HTS.

[0023] The present invention addresses the problems indicated above. In
particular, the present invention addresses the problem of providing an
efficient system allowing for rapid, dynamic and quantitative HTS of
active agents of TNFRSF members. It is in particular an objective to
provide a non-invasive and/or non-destructive method of screening, which
allows monitoring cells exposed to candidate compounds over desired time
intervals.

[0024] It is another objective to provide a way allowing the
identification of novel treatments of conditions and diseases related to
receptors of the TNFRSF and/or their ligands and/or conditions and
diseases that can be improved by acting on such receptors.

[0025] Bernard et al. (1987). Proc. Natl. Acad. Sci. USA 84, 2125-2129
disclose a chimeric receptor containing the extracellular interleukin-2
(IL-2)-binding portion of the human IL-2 receptor and the transmembrane
and intracellular domains of the human EGF receptor. This chimeric
receptor was not functional as it did not lead to autophosphorylation of
the chimeric receptor in the presence of the ligand, a feature that is
required for the release of free calcium to the cytoplasma. Moreover,
this study did not relate to drug discovery and the results of the study
would not suggest that the chimeric receptors could be useful in
screening methods. Furthermore, the IL-2 receptor is a receptor that does
not belong to the currently 29 members of the TNFRSF.

[0026] The objectives and problems as discussed above are part of the
present invention, and further objectives and solutions become apparent
from the more specific description of the invention below.

SUMMARY OF THE INVENTION

[0027] Surprisingly, the present inventors showed that artificial proteins
resulting from the fusion of a tumor necrosis factor receptor superfamily
(TNFRSF) receptor, or at least the extracellular, ligand-binding portion
thereof, with at least the intracellular, kinase portion of a receptor
tyrosine kinase (RTK) can be expressed in host cells. Surprisingly,
ligand engagement to such chimeric receptors can transduce RTK-like
signals, such as the release of free calcium to the cytoplasm, for
example. Remarkably, the generated RTK-like signal can be measured in a
dynamic, time-resolved, qualitative and quantitative manner in HTS.

[0028] According to an aspect, the invention provides a chimeric and/or
fusion polypeptide comprising:

[0029] a first part comprising an
extracellular, ligand-binding portion of a receptor A, said receptor A
being selected from TNFRSF receptors;

[0030] a second part comprising an
intracellular, signalling kinase portion of a receptor B, said receptor B
being selected from RTKs; and,

[0031] a third part comprising a
transmembrane domain.

[0032] According to an aspect, the invention provides a chimeric and/or
fusion polypeptide comprising:

[0033] a first part comprising an amino
acid sequence of an extracellular, ligand-binding portion of a receptor
A, said receptor A being selected from TNFRSF receptors;

[0034] a second
part comprising an amino acid sequence of an intracellular, signalling
kinase portion of a receptor B, said receptor B being selected from RTKs;
and,

[0035] a third part comprising an amino acid sequence of a
transmembrane domain.

[0036] According to an aspect, the present invention provides a chimeric
and/or fusion polypeptide comprising:

[0037] a first part comprising an
amino acid sequence that is taken from and/or substantially identical to
the amino acid sequence of a full-length amino acid sequence of a
receptor A or at least of an extracellular, ligand-binding portion
thereof, wherein said receptor A is selected from TNFRSF receptors;

[0038] a second part comprising an amino acid sequence taken from and/or
substantially identical to the amino acid sequence of an intracellular,
signalling kinase portion of a receptor B, said receptor B being selected
from RTKs; and,

[0039] if not comprised in said first or said second
part, between said first and second parts, a third part comprising an
amino acid sequence taken from and/or substantially identical to a
transmembrane domain, and/or,

[0040] between said first and second parts,
a part comprising an amino acid sequence taken from and/or substantially
identical to a death domain.

[0041] In an aspect, the present invention provides a chimeric polypeptide
comprising:

[0042] a first part comprising an amino acid sequence that
is substantially identical to an extracellular, ligand-binding portion of
a receptor A, said receptor A being selected from receptors of the tumor
necrosis factor receptor super family (TNFRSF);

[0043] a second part
comprising an amino acid sequence that is substantially identical to an
intracellular, signalling kinase portion of a receptor B, said receptor B
being selected from receptor tyrosine kinases (RTKs); and,

[0044] between
said first and second parts, a third part comprising an amino acid
sequence taken from and/or substantially identical to a transmembrane
domain.

[0046] an amino acid sequence that is
substantially identical to the amino acid sequence of the extracellular,
ligand binding portion of a receptor A, said receptor A being selected
from receptors of the TNFRSF,

[0047] a transmembrane domain;

[0048]
optionally, an amino acid sequence that is substantially identical to the
amino acid sequence of a death domain; and,

[0049] an amino acid sequence
that is substantially identical to the amino acid sequence of an
intracellular, signalling kinase portion of a receptor B, said receptor B
being selected from receptor tyrosine kinases (RTKs).

[0050] According to an aspect, the invention provides a chimeric and/or
fusion polypeptide comprising at least:

[0051] an extracellular,
ligand-binding portion of a TNFRSF receptor;

[0052] a transmembrane
domain, and,

[0053] an intracellular, signalling kinase portion of an
RTK.

[0054] In an aspect, the present invention provides a method of screening
active agents in general, but preferably of a receptor A selected from
TNFRSF receptors, said method comprising the steps of:

[0055] providing
cells expressing at least one nucleotide sequence encoding the chimeric
polypeptide of any one aspect of the present invention;

[0056] exposing a
candidate agent to be screened to said cells;

[0057] measuring a
physical, biological and/or chemical value that is associated with and/or
corresponds to a cellular condition of said cells; and

[0058]
determining, from the value measured in the preceding step, if said
candidate agent is an agent exerting an activity on said receptor A.

[0059] In a aspect, the present invention provides a method of screening
active agents, preferably of a receptor A selected from receptors of the
TNFRSF, said method comprising the steps of:

[0062] measuring a
physical, biological and/or chemical value that is associated with and/or
corresponds to a cellular condition of said cells; and

[0063]
determining, from the value measured in the preceding step, if said
candidate agent is an active agent of said receptor A.

[0064] In an aspect, the present invention provides method of screening
agents, which are capable of affecting the activity of a receptor A
selected from receptors of the tumor necrosis factor receptor super
family (TNFRSF), said method comprising the steps of:

[0065] providing
cells expressing at least one nucleotide sequence encoding the chimeric
polypeptide of the invention;

[0066] exposing a candidate agent to be
screened to said cells;

[0067] measuring a physical, biological and/or
chemical value that is associated with a cellular condition of said
cells; and

[0068] determining, from the value measured in the preceding
step, if said candidate agent is an agent exhibiting an activity on said
receptor A.

[0069] In further aspects, the present invention provides nucleic acids
comprising one or more nucleotide sequences encoding any one of the
chimeric polypeptides according to the invention, one or more
transcription vectors comprising one or more nucleotide sequences
encoding any one of the chimeric polypeptides according to the present
invention, cells expressing any one of the nucleotide sequences of the
invention, cells comprising one or more transcription vectors as defined
herein, cells containing any one of the chimeric polypeptides of the
invention and cells in a membrane of which is embedded any one or more of
the chimeric polypeptides of the invention.

[0070] In an aspect, the present invention provides polypeptides as
defined and/or disclosed in the present specification.

[0071] In an aspect, the present invention provides methods for preparing
polypeptides as disclosed in the present specification.

[0072] In an aspect, the present invention provides methods of screening
as defined and/or disclosed in the present specification.

[0073] In an aspect, the present invention provides the use of
polypeptides, nucleotide sequences, vectors, and cells as defined herein
in methods of screening.

[0074] The polypeptides, cells and or methods of the invention are useful
in and/or as assays for screening agents, in particular agents exerting
an activity on TNFRSF receptors.

[0075] Further aspects and preferred embodiments of the invention are
provided in the detailed description below and in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0076] In the figures,

[0077] FIG. 1a schematically represents the first step of the cloning
strategy for the preparation of a recombinant polypeptide according to a
first embodiment of the present invention, in which a fusion gene is
formed by fusing DNA encoding the full length human TNFR1 to DNA encoding
intracellular (IC) domain of mouse platelet derived growth factor
receptor (PDGFR), a RTK, thereby creating a fusion gene.

[0078] FIG. 1b schematically represents a further step of the cloning
strategy for the preparation of a recombinant polypeptide according to
the first embodiment of the present invention. In particular, the fusion
gene shown in FIG. 1a, transferred to vector pDON221, is introduced into
the vector pcDNA3.1 Hygro GW to yield the expression vector pcDNA3.1
hygro TNFR1-PDGFR.

[0079] FIG. 2 shows fluorescence intensity measured in flow cytometry of
HEK293T cells transfected with the expression vector pcDNA3.1 hygro
TNFR1-PDGFR. Due to binding of a fluorescent specific monoclonal antibody
recognizing TNFR1 to the chimeric receptor, cells expressing the chimeric
receptor according to the first embodiment of the invention (solid line)
exhibit different fluorescence than the control cells (dotted line,
staining with an unspecific monoclonal antibody of the same isotype as
the specific monoclonal antibody recognizing TNFR1). An isotype matched
control that has no specificity to any component of the cells provides
some idea of the amount of non-specific binding that one may get with the
specific antibody.

[0080] FIG. 3a is a dose response curve obtained in an HTS setting using
the Ca2+-dependent luminescence of Aequorin cells as indicator of
activity of the chimeric receptor according to the first embodiment of
the invention following administration of increasing administration of
TNF. The dose response curve is established on the basis of the
integration of the luminescence emitted in 10 minutes following TNF
administration.

[0081] FIG. 3b is a dose response curve as FIG. 3a, with the difference
that the dose response curve is established on the basis of the intensity
of the light response in dependence of applied TNF (max-min).

[0082] FIG. 4 shows individual traces of luminescent signal over time
following administration of different TNF concentrations ranging from 50
ng/ml to 100 pg/ml to the cells containing, on their surface, the
chimeric receptor according to the first embodiment of the invention. One
trace corresponds to one sample exposed to a specific concentration.

[0083] FIG. 5a shows the luminescent signal (AUC) of cells of the first
embodiment of the invention exposed to medium, TNF and TNF together with
a TNFR1-specific antibody, respectively. The antibody, binding to the
extracellular part of TNFR1, blocks TNF mediated signalling.

[0084] FIG. 5b is as FIG. 5a, with the difference that in the right column
TNF is co-administered with a PDGFR tyrosine kinase inhibitor instead of
the TNFR1-specific antibody. The signalling is blocked as in FIG. 5a,
this time due to inactivation of the tyrosine kinase activity of the
chimeric receptor of the present invention.

[0085] FIG. 6 shows dose response curves of cells of the first embodiment
of the invention (squares) and cells transfected to express the full
length PDGFR (circles) exposed to increasing concentrations of the same
inhibitor used in FIG. 5b. The cells of the invention were exposed to
TNF, whereas the other cells were exposed to human PDGF-BB.

[0086] FIG. 7 is a scatter plot showing the calcium flux or concentration
as area under the curve (AUC) of luminescence units for individual
samples containing cells of the first embodiment of the invention exposed
to medium (on the left) and to the EC80 concentration of TNF (on the
right). The indicated figure of 0.59 corresponds to the Z'-factor of the
assay, demonstrating the suitability of the assay for HTS.

[0087] FIG. 8a shows a dose response curve obtained with cells according
to a second embodiment of the invention. Cells were transfected with a
nucleotide sequence encoding a chimeric receptor comprising a truncated
TNFR1 (extracellular and transmembrane domain) fused to the cytoplasmic,
tyrosine kinase domain of a PDGFR. The light signal reflects
intracellular Ca2+ concentration, but, in contrast to the setting
underlying FIGS. 3a and 3b, is established on the basis of Fluo-4 AM, a
cell-permeable, fluorescent Ca2+ indicator.

[0088] FIG. 8b is as FIG. 8a, but obtained with cells according to a third
embodiment of the invention. Cells of this embodiment were transfected
with a nucleotide sequence encoding a chimeric receptor comprising a
truncated (only extracellular domain) TNFR1 fused to the cytoplasmic
tyrosine kinase and the transmembrane domain of a PDGFR.

[0089] FIG. 9 shows fluorescence intensity measured in flow cytometry of
HEK293T cells transfected with the expression vector pcDNA3.1 hygro
DR3(fl)-PDGFR, expressing a nucleotide sequence encoding a chimeric
polypeptide comprising the full-length DR3 receptor, in accordance with
another embodiment of the invention. DR3 is also known as TNFRSF member
25, another member of the TNFRSF. Due to binding of a fluorescent
specific monoclonal antibody recognizing DR3 to the chimeric receptor,
cells expressing the chimeric receptor according to the first embodiment
of the invention (solid line) exhibit different fluorescence than the
control cells (dotted line, staining with an unspecific monoclonal
antibody of the same isotype as the specific monoclonal antibody
recognizing DR3). An isotype matched control that has no specificity to
any component of the cells provides some idea of the amount of
non-specific binding that one may get with the specific antibody.

[0090] FIG. 10 depicts the dose response curve obtained in an HTS setting
using the Ca2+-dependent luminescence of Aequorin cells as indicator
of activity of the chimeric receptor mentioned with respect to FIG. 9
above, following administration of increasing administration of TL1A
(also known as Tumor necrosis factor ligand superfamily member 15 or
Vascular endothelial growth inhibitor). The dose response curve is
established on the basis of the integration of the luminescence emitted
in 10 minutes following TL1A administration.

[0091] FIG. 11 shows the individual traces of luminescent signal over time
following administration of different TL1A concentrations ranging from 1
ng/ml to 2 μg/ml to the cells containing, on their surface, the
chimeric receptor described with respect to FIG. 9 above. One trace
corresponds to one sample exposed to a specific concentration.

[0092] FIG. 12 depicts the dose response curve obtained in an HTS setting
using the Ca2+-dependent luminescence of Aequorin cells as indicator
of activity of the chimeric receptor FAS (extracellular and transmembrane
domains) fused to the cytoplasmic tyrosine kinase domain of PDGFR
according to a further embodiment of the invention, following
administration of increasing concentrations of FAS ligand (FASL). The
dose response curve is established on the basis of the integration of the
luminescence emitted in 17 minutes following FASL administration.

[0093] FIG. 13 is as FIG. 12, with the difference that the chimeric
receptor consists of the extracellular domain of FAS, fused to the
transmembrane and cytoplasmic tyrosine kinase domains of PDGFR according
to a further embodiment of the invention.

[0094] FIG. 14 is as FIGS. 12 and 13, with the difference that the
chimeric receptor consists of the full length FAS fused to the death
domain of TNFR1 and the cytoplasmic tyrosine kinase domain of PDGFR
according to a further embodiment of the invention.

[0095] FIG. 15 is as FIGS. 12-14, with the differences that the chimeric
receptor consists substantially of the extracellular and transmembrane
domains of the FAS receptor fused to the cytoplasmic domain of TNFR1 and
the cytoplasmic tyrosine kinase domain of PDGFR, according to a further
embodiment of the invention, and that the dose response curve is
established on the basis of the integration of the luminescence emitted
in 22 minutes following FASL administration.

[0096] FIG. 16 depicts the dose response curve obtained in an HTS setting
using the Ca2+-dependent luminescence of Aequorin cells as indicator
of activity of the chimeric receptor TNFR2 full length fused to the death
domain of TNFR1 and the cytoplasmic tyrosine kinase domain of PDGFR
according to a further embodiment of the invention, following
administration of increasing concentrations of TNF. The dose response
curve is established on the basis of the integration of the luminescence
emitted in 10 minutes following TNF administration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] The present invention provides chimeric and/or fusion polypeptides
comprising at least two parts originating from different proteins. The
chimeric polypeptide may comprise at least two amino acid sequence parts.
In particular, the chimeric polypeptide functions as a chimeric receptor.
The chimeric polypeptide may be provided in the form of a protein
isolate, but is generally provided in a cell or on the surface of a cell,
in particular embedded in a membrane of a cell, preferably in the plasma
membrane.

[0098] The chimeric polypeptide preferably comprises a first part, which
is taken from and/or substantially identical to a receptor A, or at least
a part or stretch thereof, said receptor A being preferably as defined
below. Preferably, said first part comprises an amino acid sequence part
taken from and/or substantially identical to the amino acid sequence of
said receptor A, or preferably comprising the extracellular domain of
said receptor A.

[0099] For the purpose of the present specifications, the expressions
"first part", "second part" and "third part", and "fourth part" are used.
The words "first", "second", "third" and "fourth" are, in principle not
used to express any kind of priority or relative importance of the
various parts, but are generally used to differentiate the various
structural elements of the chimeric polypeptide of the invention for
purposes of clarity. Instead of "first part", one could, for example also
use the expression "TNFRSF part", and instead of "second part", one could
use the expression "RTK-tyrosine kinase part", for example, or other
terms reflecting the function and/or origin of the respective sequence
parts. One can also omit the wording "first part", etc, altogether while
referring to the corresponding sequence stretch and/or function. With
respect to the third part, this part is only necessary as a separate part
in case one does not make use of the transmembrane domain of the TNFRSF
receptor or of the RTK receptor.

[0100] According to an embodiment, said first part comprises an amino acid
sequence that is substantially identical to the full-length amino acid
sequence of said receptor A. It is particularly surprising that chimeric
receptors comprising a full length target receptor (receptor A) and, in
addition, an intracellular portion substantially identical to the one of
an RTK (protein B) as defined below constitute a functional signal
transduction unit. This is surprising, because, without wishing to be
bound by theory, the intracellular portion of such target receptors
(receptors A) was previously thought to be obstructive to or to even
prevent activation of the intracellular portion of an RTK or at least the
transduction of RTK-like signals, due to conformational changes affecting
said intracellular part of said receptor A. In particular, one could
assume that the intracellular portion of said full length receptor A
would, upon binding of an active agent and/or ligand, move a tyrosine
kinase portion of the RTK to a spatial position or orientation were
RTK-like signals are not transduced. The inventors of the present
invention are not aware of any instance were a full-length TNFRSF
receptor was fused to a cytoplasmic tyrosine kinase domain of an RTK to
yield a functional chimeric polypeptide.

[0101] The expression "full length", according to an embodiment, does also
but not only encompasses the situation where an amino acid sequence of a
given receptor is completely and/or identically used as occurring in
nature. This term preferably also encompasses the situations that one or
more amino acids are missing or replaced, in particular functionally not
or less relevant amino acids. The expression "full length" preferably
means that all functional units of a given receptor, such as ligand
binding, transmembrane and intracellular domains, such as recruiting
domains and the like, are present. According to a preferred embodiment,
the term "full length" means in particular that there is an absence of a
truncation of one or more substantial continuous sequence portions, such
as one or more substantial portions of the cytoplasmic domain. In
particular, the expression "full length" is intended to encompass
sequences of receptors in which up to 50, preferably up to 45, 40, 35,
30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 continuous amino acid
moieties are missing if compared to the native or original receptor A.

[0102] Furthermore, the expression "full length" preferably also
encompasses situations where an artificial amino acid sequence is
provided, encoded or used, which artificial sequence combines portions of
related, similar or homologous proteins, for example as present in
different species, in a similar manner and/or in the same order of
functional entities and/or portions as they are provided in a particular
receptor A or protein B as defined herein.

[0103] According to another embodiment, said first part does not comprise
the full-length amino acid sequence, but comprises a portion, which is
taken from and/or substantially identical to a stretch of the amino acid
sequence of said receptor A. Preferably, the first part comprises an
amino sequence that is taken from and/or substantially identical to at
least a major part of the extracellular, ligand-binding portion of said
receptor A, an more preferably the complete extracellular, ligand-binding
portion of said receptor A.

[0104] The expression "a major part" includes, for the purpose of the
present specification, the situation where said first part comprises one
or more stretches that are identical to one or more stretches found in
said receptor A, so that said entire first part preferably may comprise a
continuous stretch that has at least 30%, 40%, 50% or more sequence
identity or more, as indicated elsewhere in this specification, if
aligned with the extracellular, ligand binding portion of said receptor
A.

[0105] As becomes clear from the above, said first part is preferably
defined so as to encompass any possible amino acid sequence stretch taken
from and/or substantially identical to an amino acid sequence of said
receptor A, with the proviso that it comprises at least the
extracellular, ligand-binding portion, but possibly more than that, for
example also including partially or totally the transmembrane domain of
said receptor A, and/or partially or totally the intracellular portion of
said receptor A.

[0106] According to an embodiment, said first part has any one or both of
the following capacities and/or retains any one or both of the following
functions of said original receptor A:

[0107] (a) oligomerization, for example di-, tri- and/or polymerization,
with the corresponding extracellular domain of the receptor A and/or with
the extracellular domain of another chimeric polypeptide according to the
invention;

[0108] (b) binding of an agent exhibiting an activity, for example of a
natural ligand of the receptor A.

[0109] The capacity (a) may actually be and preferably is dependent on
binding of a ligand as mentioned under (b).

[0110] Regarding the capacity (a) of oligomerization as conferred by said
first part of said receptor A it is noted that this preferably includes
the capacity of pre-ligand, dimer assembly and thus dimerization,
although such dimers are supposed not to be signalling (see publication
of (Chan, Francis Ka-Ming, Cytokine 2007, 37(2): 101-107)).

[0111] Preferably, the capacity (a) of oligomerization as conferred by
said first part of said receptor A encompasses the capacity of
trimerization, as it is thought that trimerization is seen as a common
initiating event in the TNFRSF signaling cascades (see above).
Furthermore, according to an embodiment, said capacity (a) of
oligomerization may also refer to the capacity or function of assembly of
ligand-receptor trimers into higher complexity structures (n-trimers,
hexa-, nona-, dodecamers, etc., as specified above).

[0112] In receptors A, signaling is supposed to be dependent on binding
and possibly and/or generally oligomerization, for example dimerization,
or even polymerization. Accordingly, the properties or functions (a) and
(b) may be determined by the assay as shown in the examples. In
particular, said first part may be fused to a second part, wherein said
second part is known to be functional, for example because it comprises a
functional kinase portion as specifically disclosed in Example 1. If any
first part as defined herein, if fused to said second part, is capable of
signaling if exposed to its natural ligand as demonstrated in a dose
response curve as shown, for example, in FIG. 3a or 3b and the
corresponding methodology.

[0113] In other words, in said first part, the amino acid sequence taken
from and/or substantially identical to the amino acid sequence of said
receptor A is sufficiently complete and/or identical to the corresponding
portion of said receptor A so as to confer to the chimeric polypeptide of
the invention similar and/or preferably substantially the same ligand
binding properties, ligand-binding characteristics and/or affinities as
the extracellular, ligand binding portion of said original receptor A.

[0114] Said receptor A is preferably a receptor selected from receptors of
the TNFRSF.

[0115] Table 1 below lists exemplary receptors of the TNFRSF and protein
accession numbers of receptors in the organisms indicated. Said receptor
A, may, for example, be a receptor selected from the receptors listed in
Table 1.

[0116] Preferably, receptor A is a receptor selected from type 1
(extracellular N terminus) receptors of the TNFRSF. Currently there are
29 TNFRSF members. Preferably, receptor A is selected from TNFRs, and
most preferably from TNFR1 and TNFR2.

[0117] It is particularly surprising that the chimeric polypeptide
comprising the extracellular domain of a TNFRSF receptor is suitable for
the purposes of the present invention. In vivo, receptors of the TNFRSF
are believed to exist in a pre-ligand, dimer assembly (Chan, Francis
Ka-Ming, Cytokine 2007, 37(2): 101-107). Pre-ligand dimerization is,
however, expected to activate the cytoplasmic tyrosine kinase domain of
said chimeric polypeptides and to induce RTK-like signals, since RTKs are
active as dimers. Surprisingly, however, no RTK signal is measured in the
absence of a ligand of the chimeric polypeptide and/or receptor A.

[0118] The first amino acid sequence part of said chimeric polypeptide of
the invention preferably comprises and more preferably consists of an
amino acid sequence taken from and/or substantially identical to the
amino acid sequence of said receptor A, or at least the extracellular,
ligand binding part thereof. Similar terminology is used with respect to
receptor B, discussed in more detail further below.

[0119] The expression "substantially identical to" for the purpose of the
present invention and in particular with respect to the first part of the
chimeric polypeptide, refers to amino acid sequences having at least 50%,
preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the
corresponding sequence or sequence portion or stretch (for example, the
extracellular portion) of receptor A, for example.

[0122] Sequence identity of a sequence of comparison with respect to an
original sequence is reduced when, for example, any one of the compared
or the original sequence lacks amino acid residues, has additional amino
acid residues and/or has one or more amino acid residue substituted by
another residue. Sequences having as little as 50% sequence identity with
any sequence as defined herein may still provide functional, that is,
having, independently, ligand binding functionality, tyrosine kinase
functionality, transmembrane functionality, and possibly further and/or
other functionalities as defined herein, and are thus suitable to meet
the objectives of the invention.

[0123] In the case of the extracellular, ligand-binding portion of said
first part of said chimeric polypeptide, taken from and/or substantially
identical to said receptor A, generally higher sequence identity
percentages if compared to receptor A are preferred, in order to retain
to a large extent the ligand binding and/or oligomerization properties of
the original receptor A. According to a preferred embodiment, for this
portion of the first part, there is at least 80% and more (as indicated
above) sequence identity with receptor A. With respect to transmembrane
portions and/or the intracellular portion taken of RTKs (receptor B,
discussed below), lower sequence identity levels may be sufficient to
maintain the function of the chimeric polypeptide of the invention.

[0124] According to an embodiment, "substantially identical" refers to
sequence identities of at least 80% and 60% identity of said first and
second parts with said amino acid sequence portion of said receptors A
and B, respectively, more preferably at least 85% and 70%, most
preferably at least 90% and 80%. However, sequence identities of said
first and second part may be independently selected, preferably in
dependence of the functionalities as described elsewhere in this
specification.

[0125] The chimeric polypeptide comprises a second part, which is taken
from and/or substantially identical to an intracellular, signaling kinase
portion of a receptor B, said receptor B being selected from receptor
tyrosine kinases (RTKs). Preferably, said second part is an amino acid
sequence part taken from and/or substantially identical to the amino acid
sequence of an intracellular, signaling kinase portion of a receptor B.
The expression "substantially identical" has, independently, the meaning
as detailed above.

[0126] According to an embodiment, the second part comprises the entire
intracellular portion of said receptor B.

[0127] Preferably, said receptor B is preferably selected from receptors
of the RTK super family (RTKSF). More preferably, receptor B is selected
from RTKs, which are not present in a disulfide bridged dimer in the
non-active state. RTKs of this latter type, such as the insulin receptor,
are activated by a mode of activation that is different from
ligand-induced dimerization. Preferably, the said receptor B is selected
from RTKs that are characterised by ligand-induced dimerization.

[0128] RTKs represent classical examples of surface receptors whose
activation relies upon dimerization and/or ligand-induced global
conformational changes. RTK are single-pass membrane proteins with an
extracellular ligand-binding domain and an intracellular kinase domain.
Members of this large group of membrane proteins have been classified on
the basis of their structural and ligand affinity properties (Fantl et
al. 1993 Annu. Rev. Biochem. 62, 453). The RTK family includes several
subfamilies, including the epidermal growth factor receptors (EGFRs or
ErbBs), the fibroblast growth factor receptors (FGFRs), the insulin and
the insulin-like growth factor receptors (IR and IGFR), the platelet
derived growth factor receptors (PDGFRs), the vascular endothelial growth
factor receptors (VEGFRs), the hepatocyte growth factor receptors
(HGFRs), and the nerve growth factor receptors (NGFRs) (van der Geer et
al. 1994 Annu. Rev. Cell Biol. 10, 251). The receptor B may be selected
from any one of the aforementioned RTKs. According to a preferred
embodiment, receptor B is selected from PDGFRs, EGFRs, FGFRs, and VEGFRs.
To mention a few specific examples, mouse PDGFR is available under
accession number NM--008809.1 human EGFR is available under
accession number NM--005228, human FGFR is available under accession
number NM--015850.3, human VEGFR is available under accession number
NM--002019.

[0129] Table 2 below lists receptors of the RTK super family (RTKSF). Said
receptor B may, for example, be selected from the receptors listed in
Table 2 below.

[0130] According to an embodiment, The expression "substantially identical
to" for the purpose of the present invention and in particular with
respect to the second part of the chimeric polypeptide, refers to amino
acid sequences having at least 50%, preferably at least 55%, 60%, 65%,
70%, 75%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% sequence identity with the corresponding sequence portion or
stretch of receptor B, for example.

[0131] According to an embodiment, said second part has any one or both of
the following capacities and/or retains any one or both of the following
functions of said receptor B:

[0132] (c) oligomerization, in particular dimerization, with the
corresponding intracellular domain of the receptor B and/or with the
intracellular portion of another chimeric polypeptide according to the
invention;

[0133] (d) tyrosine kinase activity.

[0134] As becomes clear from the discussion above and elsewhere in this
specification, the capacity or function of oligomerization of said
receptor B that may preferably be retained by the second part of the
chimeric polypeptide may not necessarily be or result in the same type of
oligomerization as of said first part/receptor A. In particular, in the
case of the second part, the term oligomerization preferably exclusively
refers to dimerization.

[0135] Without wishing to be bound by theory, it is also supposed that the
function or capacity of oligomerization of said second part may encompass
or even consist substantially of a type of trans-oligomerization with a
corresponding part of another individual chimeric polypeptide. Therefore,
without wishing to be bound by theory it is speculated that tyrosine
kinase activity of the chimeric polypeptide of the invention can occur as
a result of di- or oligomerization of oligomerized chimeric polypeptides.
In other words, it is possible that an oligomeric receptor complex formed
by the oligomerization of two (or three, etc.) first parts of two (or
three, etc.) chimeric polypeptides following ligand binding, needs
subsequently oligomerizing with a corresponding oligomeric receptor
complex.

[0136] In an analogous manner to the indications above with respect to
receptor A, the properties (c) and/or (d) of the second part may be
determined on the basis of the methodology as shown in the examples. If a
given second part, if combined with one of the functional first parts as
disclosed in the examples results in a chimeric polypeptide capable of
tyrosine kinase mediated signalling, said properties (c) and (d) are most
probably achieved by said second part.

[0137] In said second part, the amino acid sequence taken from and/or
substantially identical to the amino acid sequence of the intracellular,
signaling kinase portion of a receptor B is preferably sufficiently
complete and/or identical to the respective portion of said receptor B so
as to confer to the chimeric polypeptide of the invention similar and/or
preferably substantially identical RTK characteristics, such as one or
more selected from the generation of an RTK-like signal, tyrosine kinase
activity, in particular tyrosine kinase auto- and/or transphosphorylation
activity, and oligomerization with an intracellular domain of an RTK.
Without wishing to be bound by theory, it is believed that the
intracellular kinase portion, in order to transduce a signal, needs to be
capable of trans- and/or autophosphorylation. This means that two kinase
portions are in a relationship wherein the one cytoplasmic tyrosine
kinase domain phosphorylates the other and vice versa, and each one
possibly phosphorylates tyrosine residues of itself. Tyrosine
autophosphorylation is then believed to recruit and activate a variety of
signaling proteins.

[0138] The intracellular domain of RTKs generally comprises the tyrosine
kinase domain and additional regulatory sequences that are subjected to
autophosphorylation and phosphorylation by heterologous protein kinases.
According to an embodiment, said second part comprises an amino acid
sequence taken from and/or substantially identical to the tyrosine kinase
domain and also the additional regulatory sequences. Preferably, the
second part comprises at least the regulatory sequences necessary for the
generation of an RTK-like signal.

[0139] The chimeric polypeptide of the invention comprises, for example in
the form of a third part, a transmembrane domain situated between the
extracellular, ligand-binding portion of said receptor A and the
intracellular, kinase portion of said receptor B. The transmembrane
domain preferably connects and/or links said first and second parts
together. In this way, a chimeric transmembrane receptor is formed.

[0140] In principle, the transmembrane domain may be of any structure, and
may thus be selected from transmembrane domains comprising one or a
stable complex of several alpha helices, a beta barrel, a beta helix and
any other structure. According to a preferred embodiment, the
transmembrane is a single alpha helix.

[0141] Conveniently, the transmembrane domain stems from any one of the
two receptors, receptor A or receptor B. Accordingly, if the first part
of the chimeric protein comprises an amino acid sequence taken from
and/or substantially identical to the full-length amino acid sequence of
receptor A, a transmembrane domain is already present in the chimeric
polypeptide. The same is true if the first part comprises substantially
the extracellular domain and the transmembrane domain of said receptor A
but not its intracellular part (truncated receptor A). On the other hand,
the present invention encompasses the possibility that said first part
comprises only the extracellular, ligand binding part of said receptor A
(also truncated). In this case, the transmembrane domain may be selected
from any other transmembrane domain. Conveniently, the transmembrane
domain of the receptor B may be used, for example. In this case, the
second part of the chimeric polypeptide of the invention comprises, for
example, the amino acid sequence taken from and/or substantially
identical to the amino acid sequence stretching in a continuous manner
from the N-terminus of the transmembrane to the C-terminus of the
intracellular RTK domain.

[0142] As the skilled person will understand, the origin of the
transmembrane portion is generally not relevant, but it is particularly
convenient in terms of construct preparation if the chimeric polypeptide
contains a transmembrane domain of one of the two mandatory parts of the
chimeric polypeptide (TNFRSF receptor or RTK receptor) at the appropriate
position. This is, of course, because these receptors are themselves
transmembrane receptors that possess a transmembrane domain. It is thus
particularly convenient to use at least the extracellular and the
transmembrane domains of the receptor A. Accordingly, the C-terminus end
of the truncated receptor A is fused to the N-terminus of the
intracellular domain of the truncated receptor B (with or without the
intracellular, cytoplasmic domain of receptor A). Alternatively, the
transmembrane portion of receptor B is used. Accordingly, the N-terminus
of the truncated receptor B is fused to the C-terminus of the
extracellular portion of truncated receptor A. The present invention does
not exclude the possibility that the chimeric polypeptide comprises part
of the transmembrane domain of a receptor A and part of the transmembrane
domain of a receptor B, fused in such a way so as to form a "chimeric
transmembrane domain".

[0143] According to an embodiment, the part comprising the transmembrane
domain (for example, the third part) has at least 50%, preferably at
least 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% sequence identity with the transmembrane
portion of any one selected from receptors A and receptors B. In more
general terms, the transmembrane domain may be taken from or be
substantially identical to the transmembrane domain of any type 1 single
pass transmembrane receptor (e.g. cytokine receptors, receptors of the
TGFβ super family, interleukin receptors), or even other
transmembrane receptors. Preferably, the transmembrane domain is an
a-helical single pass transmembrane domain.

[0144] The transmembrane domain preferably provides the function of
anchoring the chimeric polypeptide in a membrane of cells harbouring the
chimeric polypeptide, for example cells expressing a nucleotide sequence
encoding the chimeric polypeptide. Preferably, the transmembrane domain
is suitable to keep and/or stabilise the chimeric polypeptide in the
plasma membrane of the cells.

[0145] According to an embodiment, the polypeptide of the invention
comprises one or more death domain(s). The death domain may be included
in part 1, for example, or in any other part. It is preferably located
between the transmembrane domain and the cytoplasmic portion of receptor
B (for example, part 2). The death domain may be the death domain
possibly contained in said selected receptor A. Alternatively, the death
domain may be from a different receptor, and may thus be independently be
selected (see examples below). The invention thus encompasses that the
chimeric polypeptide comprises amino acid sequence parts taken from three
different receptors. In particular, the polypeptide may comprise a
sequence part comprising an amino acid sequence taken from and/or
substantially identical to a death domain. This part may be considered a
fourth part, in particular if not contained in said first part, or
possibly in said second or third part. The function and characteristics
of death domains has been reported in the literature. Death domains form
an own protein domain super family, which is designated with accession
number c102420 and PSSM ID number 141404 at the CNBI conserved domains
database. In particular, conserved domains pfam00531 and smart00005 are
conserved domains of the death superfamily.

[0146] A death domain of a sequence will generally be recognized when the
sequence is entered at the conserved domain search mask
(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), using the defaults
settings (allowing for the low-complexity filter in the concise result
mode), with the exception of the expect value (E-value) threshold, which
may be set to 1.0, preferably 0.1, and most preferably to the default
value of 0.01. For literature see: Marchler-Bauer A et al. (2009), "CDD:
specific functional annotation with the Conserved Domain Database.",
Nucleic Acids Res.37(D)205-10.

[0147] The presence of domains, such as extracellular, transmembrane and
cytoplasmic domains, or substantially full-length sequences of receptors
of the TNFRSF and/or of receptor tyrosine kinases, including the domains
of embodiments of preferred receptors as defined herein and/or domains
thereof may also be determined using this method.

[0148] According to this method, position-specific scoring matrices
(PSSMs) derived from input "reference" sequences are used to identify
conserved domains, such as the death domain, using RPS-BLAST (Reverse
Position-Specific BLAST).

[0149] In the conserved domain database, a consensus sequence (most
frequently occurring residue at each position) of the conserved domain is
established, and, in sequence comparisons, alignment of a query sequence
with the consensus sequence is shown. The consensus sequence of the
pfam00531 death domain is:

[0150] Specifically, conserved amino acid moieties in the consensus
sequence are Gly12, Trp15, Leu18, Ala19, Arg20, Leu22, Gly23, Ile29,
Ile32, Glu33, Pro37, Ser41, Pro42, Tyr44, Leu46, Leu47, Trp50, Gln52,
Arg53, His54, Gly55, Ala58, Thr59, Leu63, Ala66, Leu67, Gly71, Arg72,
Asp74, Glu77, and Ile79 (underlined above). These amino acids at these
positions have a score of at least 5, at least 6 or higher. According to
an embodiment, a death domain in accordance with the present invention is
a sequence, when aligned with the consensus sequence as indicated above,
can be aligned with and comprises at least 2, 3, 4, 5, 6, 7, 8, 9, and
most preferably at least 10 identical amino acids of the above list of
particularly conserved amino acids. Most preferably, and possibly in
addition to the above criterion, a death domain in a sequence is present,
if, when aligned with the consensus sequence, conserves one, a selection
of two and preferably all three of Trp15, Ile29, and Trp50 of the
consensus sequence. Trp50 is the most conserved amino acid, appearing in
more than 80% of all sequences found to have a death domain.

[0151] As an example, the sequence of human TNFR1 used in for the purpose
of the present invention (SEQ. ID. NO.: 2, aa1-455), comprises a death
domain (aa359-438), and has the following amino acid moieties in common
that can be aligned with the pfam00531 consensus sequence: Leu3(359),
Ala5(361), Trp15(371), Glu17(373), Arg20(376), Leu22(378), Gly23(379),
Leu24(380), Ser25(381), Glu28(384), Ile29(385), Asp30(386), Glu33(389),
Asn36(392), Leu39(396), Arg40(397), Tyr44(401), Leu47(404), Trp50(407),
Arg53(410), Ala58(416), Thr59(417), Leu63(421), Leu67(425), Arg68(426),
Glu77(435), Ile79(437), Glu80(438). Accordingly, the death domain of
hTNFR1 has 16 identical amino acids that can be brought in alignment with
the above consensus sequence of the pfam00531 conserved domain.

[0152] Further or other death domains can be aligned with conserved domain
smart 0005. The above criteria may be independently used to determine the
presence of a death domain by examining the presence of specifically
conserved amino acid moieties with a score of at least 5 or at least 6
present in a query sequence.

[0153] According to an embodiment, the chimeric polypeptide comprises a
sequence stretch that has at least 50%, preferably at least 55%, 60%,
65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% sequence identity with the death domain of any one of
receptor A, in as far as applicable, for example TNFR1, and/or in
particular with the consensus sequence of the pfam00531 death domain
indicated above.

[0154] The death domain, if present, is a complete, that is, functional
death domain, which is capable of undergoing conformational change, for
example conformational re-orientation and/or unfolding of the stem
cc-helix (helices 5 and 6), following ligand binding. Without wishing to
be bound by theory, the present inventors believe that the presence of a
death domain may assist in the preferential orientation of the RTK domain
within the cell plasma so as to be able to be activated and to transduce
a signal.

[0155] According to an embodiment, the chimeric polypeptide comprises a
death domain that is taken from and/or substantially identical to the
death domain of the TNFR1. According to an embodiment, this applies in
particular if the chimeric polypeptide comprises a full length amino acid
sequence of a receptor A or, besides the extracellular portion of a
receptor A, the intracellular portion of a receptor A, for example
another receptor A. In this regard, according to an embodiment, a
functional polypeptide that was prepared in the examples comprises the
extracellular portion of a first receptor A (e.g. FAS) and the
cytoplasmic portion of a second receptor A (e.g. TNFR1), besides said
second part. According to this embodiment, the chimeric polypeptide
comprises a death domain of TNFR1.

[0156] According to another embodiment, a functional polypeptide that was
prepared in the examples comprises substantially the full length amino
acid sequence of a first receptor A (e.g. TNFR2) and the death domain of
TNFR1, besides said second (RTK-) part.

[0157] According to an embodiment, the chimeric polypeptide lacks a
cytoplasmic portion of a receptor A, or, in case the chimeric polypeptide
comprises a cytoplasmic portion of a receptor A, said chimeric
polypeptide preferably comprises a death domain, in particular the death
domain of TNFR1. This applies in particular if said cytoplasmic portion
of a receptor A is provided on the N-terminal side of the second part of
said chimeric polypeptide.

[0158] It is found that the present invention does also work if a death
domain is absent. In this case, however, it is preferable that the RTK
domain is situated close to the plasma membrane. Preferably, in the
chimeric polypeptide of the invention, the RTK domain follows immediately
the transmembrane domain, or is separated by a relatively short linker,
spacer or other amino acid sequence to the RTK domain. Preferably,
between the gap between the last amino acid moiety of the transmembrane
domain at the inner side of the plasma membrane and the first amino acid
of the following RTK domain spans 80 or less, preferably 70, 50, 40, 30,
20, 10, 5 or less amino acid moieties.

[0165] Amino acid moieties or sequences having or, independently, not
having further functionalities, may or may not, independently, be
provided terminally and in positions indicated with "-".

[0166] According to an embodiment, the encoded TNFRSF domains and the
encoded RTK as shown, for example, under no. 1-6 above, are linked (for
example, functionally or structurally linked or joined), for example
translationally linked, for example as a fusion protein.

[0167] According to an embodiment, the chimeric polypeptide of the
invention is a chimeric transmembrane protein, preferably a chimeric
transmembrane receptor. Preferably, the chimeric polypeptide has an
extracellular N-terminus and an intracellular C-terminus. In the list
above (no. 1-6), the elements of the chimeric polypeptide are thus
preferably shown from the N terminus (left) to the C-terminus (right).

[0168] Preferably, the individual parts of different origin of the
chimeric polypeptide, when embedded in the plasma membrane of cells, are
provided in the same position and/or substantial orientation as in the
original protein from which sequence parts were taken. Accordingly, the
chimeric polypeptide is a type 1 single pass transmembrane receptor.
Preferably, the N- and C-termini of the sequence stretch that
substantially corresponds to the intracellular sequence of a receptor B
correspond to the corresponding termini and/or orientation as found in
the original receptor B. The same applies in analogy to sequences that
are substantially identical to sequences of a receptor A. Preferably,
only one transmembrane domain is present, which preferably separates the
intracellular parts from extracellular parts of both original receptors A
and B. In other words, the transmembrane domain is positioned
appropriately. For example, if the chimeric receptor also comprises the
intracellular part of a receptor A, the transmembrane domain is located
on the amino acid sequence so that also in the chimeric polypeptide the
intracellular part of receptor A is on the intracellular side of the
chimeric polypeptide.

[0169] The reference receptors A and B are preferably of a natural origin.
They may be as already reported, or they may be receptors that still will
be discovered in the future, and to which the principle of the present
invention can be applied. Of course, receptor A is selected in dependence
of the purpose of the screening method, that is, the target, for which an
active agent is sought. Accordingly, receptor A and receptor B may
independently be isolated from any organism, in particular animals or
humans. Preferably, the receptors A and B are, independently, human, or
mammal animal receptors. According to an embodiment, receptors A and B
are independently as present in a human, simian, rodent, ungulate,
carnivore, bird, reptile, amphibian and/or insect. Receptors found in
humans, rodents and domesticated animals, such as pets and livestock are
preferred.

[0170] The chimeric polypeptide of the present invention thus comprises at
least stretches (or, for example in case of the first part, a full length
receptor A) of a naturally occurring receptor, or comprises sequence
stretches which may be composed of stretches of different naturally
occurring receptors.

[0171] Receptors A and B may also be referred to as "reference receptors"
or "original receptor", because, preferably, the respective part of the
chimeric polypeptide of the invention stems from and/or is substantially
identical to at least a portion of a naturally occurring receptor and the
latter is thus the basis of a comparison. However, as mentioned above, in
the amino acid sequences (and the encoding nucleotide sequences) of the
invention, the original sequences may be modified for any particular
purpose, in order to provide variants or sequences with similarity to the
original reference receptor, depending on the desired properties of the
final polypeptide.

[0172] The transmembrane domain, and the nucleotide sequence encoding it,
may again be of any origin, that is, isolated from any organism having
transmembrane protein domains, for example the organisms mentioned above.
Furthermore, natural or artificial variants may be used.

[0173] According to an embodiment, the amino acid sequence of said first
part is taken from and/or substantially identical to a continuous stretch
of at least 80, 100, 120, 150, 170, 190 and most preferably at least 200
continuous amino acid moieties of the amino acid sequence of said
receptor A.

[0174] According to an embodiment, the amino acid sequence of said second
part is taken from and/or substantially identical to a continuous stretch
of at least 200, 250, 350, 400, 450, 470, 500, and most preferably at
least 520 continuous amino acid moieties of the amino acid sequence of
said receptor B.

[0175] In other words, the compared sequences (first part to receptor A;
second part to receptor B) encompass at least one continuous stretch
preferably having at least the above indicated preferred lengths.

[0176] The chimeric polypeptide of the present invention may comprise
further amino acid sequences or may be further modified, for example in
vivo and/or in vitro, for example by chemical modification. For example
linker sequences, cell-compartment targeting sequences, sequences with
protease cleavage sites, marker sequences, oligomerization domains,
effector protein binding domains, domains assisting in protein isolation,
catalytically active domains, glycosylation, just to mention a few, may
be present on or be part of the chimeric polypeptide of the invention.
Additional amino acid sequences may be provided terminally or between
other sequence parts constituting the chimeric polypeptide of the
invention. This applies, for example, to possible linker sequences. Said
additional amino acids and/or amino acid sequences may be present also in
the embodiments numbered 1-4 above. The additional domains or sequences
may be encoded, for example, by continuous reading frame of the
nucleotide sequence encoding the chimeric polypeptide of the invention
and may or may not be removed in vitro, or, in vivo, for example by
pre-mRNA cleaving, RNA splicing, posttranscriptional modifications,
protein modification by protein splicing, proprotein convertase and
signal peptide peptidase, for example.

[0177] The chimeric polypeptide of the invention may be substantially
formed by a continuous amino acid sequence, in which each amino acid
residue is connected to the respective neighbour(s) by a peptide bond (a
fusion protein). The separate domains may, of course, contain additional
amino acid sequences as mentioned above (linkers, etc.).

[0178] Alternatively, the chimeric polypeptide of the invention may
comprise two or more separate amino acid sequences forming separate
protein domains, which may be connected covalently or non-covalently, to
form a complex comprising separate protein units. For example, one, two
or all three individual parts and/or domains of the chimeric polypeptide
(extracellular, transmembrane and cytoplasmic domains) may be connected
to the respective neighbouring domain by way of one or more disulfide
bonds.

[0179] The present invention provides one or more nucleotide sequences
encoding the chimeric polypeptide of the present invention. According to
an embodiment, the present invention provides a nucleic acid comprising a
single continuous or several separate nucleotide sequences encoding the
chimeric polypeptide of the invention. Preferably, the nucleic acid
molecule may comprise a first sequence encoding at least the
extracellular, ligand-binding portion of a receptor A, a second sequence
encoding at least the intracellular, signaling kinase portion of a
receptor B and, if not yet comprised in between said first and second
sequences, a third sequence encoding a transmembrane domain. Preferably,
said first, second and, optionally, third sequences are provided in the
form of an overall continuous coding sequence. As indicated above, the
continuous coding sequence may also encompass and/or encode further amino
acids or sequences, as exemplified elsewhere in this specification. The
nucleic acid may further comprise a promoter sequence, such as one of
those specified in more detail below, which controls expression of said
the sequence(s) encoding said chimeric polypeptide.

[0180] The attached sequence listing discloses nucleotide and amino acid
sequences, respectively, of the following exemplary fusion proteins in
accordance with various preferred embodiments of the present invention:

[0181] The fusion of full length hTNFR1 with the truncated, cytoplasmic,
tyrosine kinase domain of mouse mPDGFR: SEQ. ID. NO.: 1 and 2.

[0194] When embedded in a membrane, preferably the plasma membrane, of a
cell and under physiological conditions, the chimeric polypeptide of the
invention as disclosed and described above is preferably capable of
binding, preferably on the outer cell surface, a ligand that under
natural and/or physiological conditions binds to receptor A. Following
binding, the chimeric polypeptide is preferably capable of generating an
RTK-like or tyrosine kinase mediated signal inside the cell.

[0195] Without wishing to be bound by theory, if the receptor A is
selected from receptors of the TNFRSF, for example from TNFRs, the
chimeric polypeptide is supposed to oligomerize, preferably trimerize,
and to induce tyrosine kinase trans- and/or autophosphorylation and to
thereby induce RTK-mediated signalling. When there is ligand binding
and/or RTK-mediated signalling the chimeric receptor is in an active
condition, which is different from the condition when there is no ligand
binding, for example.

[0196] Cellular conditions affected by RTK-signalling may be recognised in
screening methods and enable thus the detection of a binding and/or
activation of the chimeric receptor of the invention. Since at least the
extracellular, binding portion of the chimeric receptor is taken from
and/or substantially identical to at least the extracellular, binding
portion of a TNFRSF receptor, any compound binding to the receptor of the
invention can be expected to be active on the original receptor (receptor
A).

[0197] According to an embodiment, said cellular condition is at least
partly dependent on an activity of said chimeric polypeptide. The
chimeric polypeptide may thus exist in an active form and in inactive
form. Furthermore, in cells containing several chimeric receptors, some
of the receptors may be active and others inactive, in particular in
dependence of the concentration of an active agent. The latter situation
results in a partial activity, so that the screening method is preferably
suitable to quantify activity on a substantially continuous scale.

[0198] Preferably, the "activity" of the chimeric polypeptide is a
signalling activity, which is generally the consequence of ligand binding
and the oligomerization of receptor subunits as discussed elsewhere in
this specification. The oligomerization following ligand binding results
in activation of tyrosine kinase activity, which in turn affects the
cellular condition.

[0199] According to an embodiment, an agent affects the activity of a
receptor if it affects a status of signalling of the receptor. The
"status of signalling" preferably refers to the presence, absence or
degree of signalling activity, of a receptor, for example all receptors
of the same type of a cell. For example, an agent is active if it stops a
receptor that is signalling, or if it induces signalling of a receptor
that was not signalling before. The term "signalling" is understood as
transducing or transmitting any kind of cellular signal to the
intracellular and/or cytoplasmic part of the cell. As the skilled person
understands, a signal may involve a cascade of intracellular and
molecular events, in particular chemical reaction, which result in the
change of the cellular condition of the cell. In particular, the
concentration of second messengers or other cellular components may
change.

[0200] Preferably, an activity of the chimeric receptor is thus equivalent
to tyrosine kinase activity, preferably as specified elsewhere in this
specification.

[0201] The present invention provides a method of screening compounds
and/or compositions of matter exhibiting and/or exerting an activity, in
particular a biological activity, on a receptor, in particular a receptor
A as defined herein. For the purpose of this specification, this is
equivalent to saying that the invention provides a method of screening
for (or of) agents that affect the activity of a receptor A. Such
compounds and/or compositions of matter may be referred to herein as
"active agents", or simply "agents". Preferably, activity refers to cell
signalling activity. A "candidate agent" may be any substance of matter.
For example, isolated chemical compounds (molecules) or compositions of
matter, such as composition of compounds, for example extracts, such as
reaction mixtures, plant extracts and the like. The compound may be a
macromolecule. In principle, the only limitation with respect to the
"agent to be screened" one can spontaneously think of is that it can be
added to a well plate of a microtiter plate comprising the cells.

[0202] Active agents, as understood in this specification, encompass and
preferably are agonists, antagonists and modulators, for example. The
agents may be binding to orthosteric and/or allosteric sites of receptor
A and/or the polypeptide of the invention. The terms agonists and
antagonists encompass natural ligands--endogenous (ant)agonists--as well
as exogenous (ant)agonists.

[0203] Modulators are generally compounds that act in a modulating manner
in conjunction with an agonists or antagonist, in particular with a
natural ligand. Modulators may again be classified as "active
modulators", which encompass and preferably consist of "inhibitors",
"activators" and/or "neutral modulators" of receptor A. "Neutral
modulators" are chemical entities that bind to the target without direct
modulation of its function, but they prevent the binding of the natural
ligand and/or other modulators or bioactive principles that share the
same binding site on the target receptor, and in that way indirectly
affect its activity and/or modulation.

[0204] According to an embodiment, the invention provides a method for
screening active agents of a receptor A selected from receptors of the
TNFRSF.

[0205] For example, if receptor A is selected from receptors of the TNFRs,
an active agent may be an agent that prevents binding of the
corresponding TNF. Such an active agent can then be used to prevent TNFR
mediated signalling.

[0206] According to an embodiment, an agent affects the activity of a
receptor if it affects a status of signalling of the receptor. The
"status of signalling" preferably refers to the presence, absence or
degree of signalling activity, of a receptor, for example all receptors
of the same type of a cell. For example, an agent is active if it stops a
receptor that is signalling, or if it induces signalling of a receptor
that was not signalling before. The term "signalling" is understood as
transducing or transmitting any kind of cellular signal to the
intracellular and/or cytoplasmic part of the cell. As the skilled person
understands, a signal may involve a cascade of intracellular and
molecular events, in particular chemical reaction, which result in the
change of the cellular condition of the cell. In particular, the
concentration of second messengers or other cellular components may
change.

[0207] In the screening method of the invention, an automated apparatus
system is preferably used. Such a system may allow one or more or all of
the following: high throughput screening; analysis of host cells
containing reporter molecules (for example, fluorescent or luminescence
reporter molecules); treating the host cells with one or more candidate
agents; treating the host cells with one or more agents of known
activity, such as the natural ligand; imaging and recording numerous
cells at once, for example with fluorescence or luminescence optics;
converting the optical information into digital data; utilizing the
digital data to determine the concentration, and/or the activity of the
reporter molecules in the cells and/or the distribution of the cells; and
interpreting that information in terms of a positive, negative or null
effect of the candidate agent on the at least one cellular
characteristic.

[0208] The screening methods of the invention preferably use cells
containing, preferably embedded in a membrane, the chimeric polypeptide,
and/or expressing a nucleotide sequence encoding the chimeric polypeptide
of the invention. These cells are also referred to as host cells.

[0209] The cells may for example be a mammalian cell such as for example a
cell of bovine, porcine, rodent, monkey or human origin. The mammalian
cell may for example be any one of the group consisting of a HeLa cell, a
U2OS cell, a Chinese hamster ovary (CHO) cell, a CHO-KL cell, a HEK293
cell, a HEK293T cell, an NSO cell, a CV-1 cell, an L-M(TK-) cell, an L-M
cell, a Saos-2 cell, a 293-T cell, a BCP-1 cell, a Raji cell, an NIH/3T3
cell, a C127I cell, a BS-C-1 cell, an MRC-5 cell, a T2 cell, a C3H10T1/2
cell, a CPAE cell, a BHK-21 cell, a COS cell (for example, a COS-1 cell
or a COS-7 cell), a Hep G2 cell, and an A-549 cell. Such cells and other
suitable cells are publicly available, for example from commercial
sources such as the American Type Culture Collection (ATCC), the European
Collection of Cell Cultures (ECACC) and/or the Riken Cell Bank (Tokyo,
Japan).

[0210] The cells may comprise and/or be transfected to express an
expression vector comprising any one of the nucleic acids and/or
nucleotide sequences as disclosed herein. Expression of the nucleic acid
may be driven by a constitutive or inducible promoter. Typically, the
promoter is positioned upstream of the nucleic acid/nucleotide sequence
encoding the polypeptide to allow transient or stable expression, for
example in mammalian cells. The expression vector may comprise a
Tet-ON® inducible expression system. Use of an inducible expression
system allows higher levels of the polypeptide of the invention to be
present when desired or required. Expression may be inducible for example
upon addition of doxycyclin, tetracycline, or an analogue of either, such
in a mammalian cell for example a CHO cell or other cells disclosed
herein. The nucleic acid/nucleotide sequence, expression vector or
polypeptide may be transiently or stably transfected into the host cell.

[0211] The cells are preferably provided at an approximately determined
number in the wells of a microtiter plate. Each well and the cells plated
therein thus constitute a sample. Cells may be added or plated in the
wells of a microtiter plate in an automated manner.

[0212] The screening method of the invention comprises the step of
exposing a candidate agent to be screened to said cell. As mentioned
above, this may be done in an automated manner. Preferably, the present
invention provides the step of adding said candidate agent at different
concentrations to different wells of a microtiter plate, preferably in an
automated manner.

[0213] The screening method of the invention comprises the step of
measuring a physical, biological and/or chemical value that is associated
with and/or corresponds to a cellular condition of said cells. Said
cellular condition is preferably an intracellular condition.

[0214] Preferably, said cellular condition is affected if said candidate
agent is an active agent, in particular of said receptor A. According to
an embodiment, said cellular condition is at least partly dependent of
and/or affected by an activity and/or condition of said chimeric
polypeptide. For example, said cellular condition is dependent on and/or
affected by the presence or absence of a specific form of oligomerization
of the intracellular and/or extracellular components of said chimeric
polypeptide, and/or for example on the RTK-activity of the intracellular
domains of the chimeric receptor, and/or of ligand binding at the
extracellular portion of the chimeric receptor.

[0215] According to an embodiment, binding of an active agent to said
chimeric polypeptide may at least to some extent induce and/or prevent
oligomerization of a plurality of said chimeric polypeptides and/or
wherein said oligomerization induces a kinase activity of said
intracellular kinase portion of the chimeric polypeptide.

[0216] According to an embodiment, the method of screening further
comprises the steps of exposing said cells to a control agent. The
control agent preferably has a known, reported and/or established effect
on the activity of said receptor A. The method preferably comprises
determining the capacity of said candidate agent to modulate activation
and/or binding of said control agent to said chimeric polypeptide.
Preferably, a candidate agent affects the activity of said receptor A if
it affects an effect of said control agent on the activity of said
chimeric polypeptide. Examples of such active agents are allosteric
modulators, such as positive or negative allosteric modulators (PAMs and
NAMs).

[0217] The control agent may be selected from orthosterically or
allosterically binding ligands of receptor A. For example, the control
agent is selected from natural ligand(s) of the receptor A. The control
agent is an agent whose concentration-response curve is reported or can
conveniently be established by the screening method of the invention, in
particular by adding different (e.g. increasing) concentrations of the
agent to the cells and measuring the intensity of the physical,
biological and/or chemical value. In this way, EC values can be
established for the control agent (ECO-EC100), indicating the minimum
concentrations to obtain a signal that is distinguishable from baseline
and the concentration that is needed to obtain a maximum signal/value.
The control agent may be added at concentrations corresponding to EC
values that are covered by the ranges EC5-100, EC5-97, EC10-90, EC20-80,
for example. Accordingly, the method of the invention may be used to
screen for modulators, which do not directly activate or inhibit a
receptor, but which modulate the receptor activity in response to a
directly activating or inhibiting agent, such as a natural ligand.

[0218] For example, the control agent (for example, the natural ligand or
the ligand of reported effect) may be added in two- or more addition
protocol, for example a co-addition protocol. In this way, inhibitors or
activators of receptor A may be found, for example.

[0219] According to an embodiment, the method of the invention comprises
the step of measuring a physical, biological and/or chemical value that
is associated with a cellular condition of said cells.

[0220] According to a preferred embodiment, said cellular condition is
affected by the activity and/or absence of activity of the intracellular
kinase domain of said chimeric polypeptide. According to an embodiment,
said cellular condition is at least partly dependent on the presence of
activity, absence of activity, and/or extent of activity of the
intracellular kinase domain of said chimeric polypeptide. As mentioned
above, said tyrosine kinase activity may, in turn, be dependent on the
binding of an active agent and/or oligomerization or absence of
oligomerization of the extracellular and/or intracellular domains of the
chimeric polypeptide.

[0221] According to an embodiment, said physical, biological and/or
chemical value that is associated with and/or corresponds to a cellular
condition is fluorescence and/or luminescence, in particular
bioluminescence.

[0222] It is noted that the expression "physical, biological and/or
chemical value" refers to any measurable signal produced by the cells
following binding and/or modulation of receptor activity. Presently, many
reporting systems produce light, which can be conveniently detected using
appropriate equipment. Light produced by a reporting system may be
produced by a luminescent protein, possibly under consumption of a
particular chemical substrate that is specifically added to the cells. In
this regard, the light amount is indeed all of the above: a physical
value (light intensity), a biological value (reflecting bioluminescent
activity) and a chemical value (reflecting substrate consumption).

[0223] One could also measure other parameters or signals, as reporting
systems producing radioactivity (less frequently used today) or other
markers (substrate consumption, product generation, etc.). The
quantification of such signals can generally in all cases be considered
as the measurement of a physical, biological and/or chemical value.
Measurements are generally made with the corresponding equipment.

[0224] Preferably, a reporting system produces a signal in dependence of a
cellular condition, such as the concentration of a cellular component,
for example a second messenger.

[0225] Preferably, the cellular condition is an intracellular condition.

[0226] Activated tyrosine kinase domains of RTKs, one of which is
substantially part of the chimeric polypeptide, are reported to be
phosphorylated or active on a variety of signaling proteins, and,
depending on the specific signal transduction pathway induced, to lead to
the recruitment of adapter, or to the release of intracellular secondary
messengers, such as Ca2+, inositol phosphate (IP1) and inositol
triphosphate (IP3). Therefore, according to an embodiment, the
intracellular condition is concentration or a change in the concentration
of one or more selected from: free intracellular Ca2+, inositol
phosphate (IP1) and inositol triphosphate (IP3). According to an
embodiment, said cellular condition is the degree in phosphorylation or
recruitment of adapter proteins.

[0229] For example, aequorin is a photoprotein isolated from luminescent
jellyfish and is composed of two distinct units, the apoprotein
apoaequorin and coelenterazine, a luciferin. The two components of
aequorin reconstitute spontaneously, forming the functional protein. The
protein bears several binding sites for Ca2+ ions, which, when
bound, trigger the protein to undergo a conformational change. As the
excited protein relaxes to the ground state, blue light (wavelength=469
nm) is emitted. Therefore, according to an embodiment, the cells of the
present invention preferably express apoaequorin. For example, the cells
are transfected to express apoaequorin. In this case, the screening
method of the invention preferably comprises the step of adding a
luciferin, in particular coelenterazine to the cells. In this embodiment,
the light emitted by aequorin (luminescence) constitutes the physical
value that is measured in the method of the invention. More specifically,
said physical value is bioluminescent light having a wavelength having a
maximum intensity in the wavelength range of 400-540 nm, preferably
440-500 nm, most preferably about 460-480 nm. Aequorin emits blue light
(wavelength=469).

[0230] The skilled person may, of course, select any other indicator of
intracellular Ca2+ concentration, such as for example, the Fluo-4 No
Wash (NW) dye mix commercially obtainable from Molecular Probes, USA. In
this and other systems, intensity and/or wavelength of fluorescent light
is dependent on intracellular free Ca2+ concentration, said
fluorescent light thus forming a measurable and interpretable physical
value.

[0231] The expressions "associated with" and/or "corresponding to" for the
purpose of the present specification have their general meaning. They
thus reflect any kind of correlation and/or link between the cellular
condition and the physical, biological and/or chemical value that can be
measured. The strength of the signal is generally associated with (that
means correlates in some way with) the cellular condition (e.g. second
messenger concentration). For example, in the case of light produced by
aequorin, the intensity of the light correlates with intracellular, free
Ca2+ concentration, so that the measurement of a light intensity can
be interpreted as a particular, approximate concentration of free
Ca2+.

[0232] The method of the invention preferably comprises the step of
determining, from the value measured in the preceding step, if said
candidate agent is an agent exhibiting an activity on said receptor A. In
this regard, the determination step generally involves the comparison of
the value of the actually measured physical, biological and/or chemical
signal in accordance with the method of the invention to a basic value.
The basic value is determined, for example, in the absence of said
candidate agent (the negative control). The basic or negative control
value may be determined beforehand, that is, before running the method of
the invention. In FIGS. 8a and 8b, the very left side, an isolated data
point in the graphs corresponds to such a basal or basic value.
Generally, a threshold value is generated or determined, which is
sufficiently far away from the negative control value so as to account
for natural variations occurring in the signal measurement. The methods
of determining such threshold values, which also relates to the avoidance
of false positives, can be established by the person skilled in the art.
The same applies with respect to the statistics that one may use to
increase the probability that a given measured deviation from the
negative control or from the threshold value corresponds indeed to a
"hit" (an active agent). In particular, measurements may be repeated and
the mean of several separate measurements may be used for purpose of
comparison and thus, determining if an agent is considered as an active
agent.

[0233] From the above it becomes clear that the administration of a
candidate agent, if it affects the cellular condition of the cells,
should induce the reporting system to produce a detectable change of the
physical, biological and/or chemical value. The candidate agent is then
considered an active agent (a hit). In accordance with an embodiment,
said candidate is an active agent of said receptor A, if it affects said
cellular condition of said cells.

[0234] The invention is disclosed in further detail in the following
examples, which are in no way intended to limit the scope of the present
invention.

EXAMPLES

Examples 1-3

Preparation of Constructs and Transfection Vectors of Chimeric TNFR1-PDGFR
in Accordance with Embodiments of the Invention

[0236] For preparing these constructs and expression vectors, standard
cloning techniques were used according to manufacturer's instructions.

[0237] The resulting PCR product encoding the chimeric receptor was
inserted into the pDONR221 vector of Invitrogen using the Gateway BP
Clonase® enzyme mix (Invitrogen), according to the manufacturer's
protocol.

[0238] To generate the appropriate expression vector the Gateway
cassette® (Invitrogen) was inserted into the ECORV site of the
pcDNA3.1 hygromycin vector (Invitrogen), using standard cloning
techniques. The chimeric receptor DNA was introduced into the expression
vector pcDNA3.1 hygro GW using the LR Clonase® II enzyme mix of
Invitrogen (FIG. 1b), according to the manufacturer's protocol, yielding
the expression construct pcDNA3.1 hygro TNFR1(fl)-PDGFR(cd) vector.

Example 4

Transfection of HEK293T Aequorin Cells and Expression of the Chimeric
Receptors

[0239] HEK293T stably expressing Apoaequorin were generated using standard
cloning techniques. The HEK293T cells expressing Apoaequorin ("Aequorin
cells") were then further transfected as described in Examples 1-3 so as
to express the chimeric receptors 1-3 as listed in Table 3.

[0240] In particular, the HEK293T Apoaequorin cells were transfected with
pcDNA3.1 hygro TNFR1-fl-PDGFR-cd vector as prepared in Example 1 using
Optifect® Transfection Reagent (Invitrogen), according to the
manufacturer's protocol.

[0241] Cell surface expression of the chimeric receptors comprising full
length TNFR1 and the cytoplasmic domain of PDGFR was detected by flow
cytometry. Briefly, cells were harvested and incubated with a monoclonal
antibody directed against TNFR1 (MAB225, R&D Systems) or an isotype
matched mouse IgG (both purchased from R&D systems, Minneapolis, Minn.,
USA). Both antibodies were used at a final concentration of 1 μg/ml.
Cells were washed twice and incubated with Cy3-conjugated F(ab')
fragments of a donkey anti-mouse polyclonal antibody (Jackson
ImmunoResearch, Westgrove, Pa., USA) at a final concentration of 0.2
μg/ml. Subsequently, cells were washed twice and resuspended in a
final volume of 500 μl. All antibody incubations were performed in
flow cytometry buffer (PBS containing 5% FBS and 0.01% sodium azide) for
20 minutes at 4° C. Flow cytometry was performed using a
FACSCalibur and results were analyzed using Cellquest software (BD
Biosciences, San Jose, Calif.).

[0242] The flow-cytometrical results are shown in FIG. 2, where the black
solid line corresponds to anti-TNFR1 mAb staining and the dotted line
corresponds to the values obtained with the isotype control.

[0243] These results show that the extracellular domain of TNFR1 of the
chimeric receptor is found at the surface of the transfected HEK293T
Aequorin cells.

Example 5

Detection of Intracellular Calcium Levels in an HTS Setting

[0244] The property of aequorin to produce light in dependence of
intracellular free Ca2+ ions is described above.

[0245] HEK293T cells expressing Apoaequorin and the chimeric receptor
(Example 4) were plated in 384-well plates at a concentration of 12500
cells per well in a final volume of 50 μl. The next day culture
supernatants were removed and 25 μl labeling buffer (DMEM:F12 plus
0.1% BSA) containing 2.5 μM Coelenterazine h (Dalton Pharma services),
was added. Cells were incubated at room temperature for 6 hrs. A FDSS7000
reader from Hamamatsu (Japan) was used to examine intracellular calcium
levels. This instrument is designed for high throughput screening and
high throughput analysis. The instrument features include detection with
a camera of fluorescence or luminescence and automatically converts
fluorescence or luminescence signals into numeric data. This digital data
is then used to determine the concentration of calcium inside the
analyzed cells. The information is automatically analyzed in terms of a
positive, negative or null effect of each test compound being examined.
This system allowed differences between untreated and treated cells to be
measured, for example by measuring the calcium flux in cells.

[0246] After a baseline reading of 10 seconds, cells were incubated for 3
minutes with different doses of inhibitors or buffer controls.
Subsequently, cells were stimulated with TNF and measurements were
continued for another 10 minutes. The results were analyzed using the
FDSS analysis software from Hamamatsu.

[0247] FIGS. 3a and 3b are dose response curves obtained by exposing the
cells of Example 4 to increasing concentrations of TNF. FIG. 3a is
established on the basis of the integration of the luminescence emitted
in 10 minutes following administration of TNF (exposure time) in
dependence of the applied TNF concentration (AUC), while FIG. 3b is
established on the basis of intensity of the response in dependence of
the applied TNF concentration (max-min). The concentration of TNF ranged
from 50 ng/ml to 100 pg/ml. The results are representative of four
independent experiments and the error bars represent the standard
deviation of triplicate wells. FIG. 4 shows the individual traces of
luminescent signal corresponding to the TNF concentrations ranging from
50 ng/ml to 100 pg/ml.

[0248] Table 4 below shows the EC50 and EC80 values determined on the
basis of the results shown in FIGS. 3a and 3b.

Effect of TNFR1 and PDGFR Agents on the Calcium-Dependent Luminescence
Signal in the Cells of the Invention in HTS

[0249] HEK293T Aequorin cells expressing fusion proteins as described in
Example 4 were treated or not treated with 3 ng/ml of TNF and 300 ng/ml
of an anti-TNFR1 antagonist monoclonal antibody (MAB225, R&D Systems) was
added to half of the samples exposed to TNF. Following 10 minutes of
exposure after TNF addition, the area under the curve was determined for
each sample. The result is seen in FIG. 5a. As can be seen, the
anti-TNFR1 antibody completely prevented TNF-induced signaling, that is,
the increase of intracellular free Ca2+. This experiment shows that
the constructs, chimeric polypeptides and cells of the invention are
suitable in screening methods of agents exhibiting an activity on TNF
receptors.

[0250] In another experiment, Aequorin cells of Example 4 exposed to 3
ng/ml of TNF were or were not exposed, besides TNF, to
4-(6,7-Dimethoxy-4-quinazolinyl)-N-(4-phenoxyphenyl)-1-piperazinecarboxam-
ide, a PDGFR Tyrosine Kinase Inhibitor III of Calbiochem (USA). As can be
seen from FIG. 5b, addition of 1 μM of inhibitor prevented the
detection of intracellular calcium increase, showing that the
TNF-dependent signal is mediated by the kinase domain of the chimeric
receptor.

[0251] FIG. 6 shows that the PDGFR inhibitor as described above has equal
inhibitory efficacy of Aequorin cells expressing the full-length PDGFR
receptor and the TNFR1-PDGFR chimeric receptor. The concentration of
PDGFR kinase inhibitor ranged from 3 μM to 0.45 nM. The results are
representative of four independent experiments and the error bars
represent the standard deviation of triplicate wells.

Example 7

Determination of Suitability for HTS

[0252] The "Z'-factor" of an assay is a statistical measure used to
evaluate a high-throughput screening (HTS) assay. A score close to 1
indicates an assay is ideal for HTS and a score less than 0 indicates an
assay to be of little use for HTS (see Zhang et al., 1999, J. Biomol.
Screen. 4: 67-73). Four parameters needed to calculate the Z'-factor are:
mean (μ) and standard deviation (σ) of both positive (p) and
negative (n) control data (μp, σp, μn,
σn, respectively). Using the formula:

Z'-factor=1-[3×(σp+σn)/|μp-μn|]

[0253] In order to determine the Z'-factor of the assay of the present
invention, the cells of Example 4 above were plated in a 384-well plate
as described above and exposed to EC80 of TNF (3 ng/mL) or to cell medium
devoid of TNF ("Media"), and the area of curve was determined following
10 minutes of exposure. FIG. 7 is a scatter plot showing the calcium flux
or concentration as area under the curve (AUC) of luminescence units for
each sample. The Z'-factor for the assay results shown in FIG. 7 was
calculated to be 0.59. The Z'-factor calculation demonstrated that the
method of the invention is validated for use in HTS.

[0255] Intracellular calcium levels were determined using the Fluo-4 No
Wash (NW) dye mix according to the manufacturer's recommendation
(Molecular Probes, USA). In short, HEK293T cells transfected as described
in Examples 1-3 so as to express the chimeric receptors 1-3 as listed in
Table 3 were plated in 384-well plates at a concentration of 12500 cells
per well in a final volume of 50 μl. The next day, culture
supernatants were removed and 25 μl labeling buffer (1×HBSS, 20
mM Hepes), containing the Fluo-4NW dye mix and 2.5 mM probenecid, was
added. Cells were incubated at 37° C. for 30 minutes, followed by
30 minutes at room temperature. Intracellular calcium levels were
determined using a FLIPR Tetra (Molecular Devices, USA). After a baseline
reading of 10 seconds, cells were stimulated with TNF and measurements
were continued for another 10 minutes. The results were analyzed using
the Screenworks software from Molecular Devices.

[0256] FIG. 8a shows a dose response curve of construct 2 established on
the basis of the intensity of the fluorescent response (max-min) in
dependence of the applied TNF concentration. The concentration of TNF
ranged from 5 μg/ml to 4 ng/ml. The results are representative of four
independent experiments and the error bars represent the standard
deviation of triplicate wells.

[0257] FIG. 8b shows a dose response curve of construct 3 established on
the basis of the intensity of the fluorescent response (max-min) in
dependence of the applied TNF concentration. The concentration of TNF
ranged from 5 μg/ml to 4 ng/ml. The results are representative of four
independent experiments and the error bars represent the standard
deviation of triplicate wells.

Examples 9-10

Preparation of Constructs and Transfection Vectors of Chimeric TNFR1-EGFR
in Accordance with Embodiments of the Invention

[0259] For preparing these constructsand, expression vectors, standard
cloning techniques were used according to manufacturer's instructions.

[0260] The resulting PCR product encoding the chimeric receptor was
inserted into the pDONR221 vector of Invitrogen using the Gateway® BP
Clonase® enzyme mix (Invitrogen), according to the manufacturer's
protocol.

[0261] To generate the appropriate expression vector the Gateway cassette
(Invitrogen) was inserted into the ECORV site of the pcDNA3.1 Hygro
vector (Invitrogen). The chimeric receptor DNA was introduced into the
expression vector pcDNA3.1 Hygro GW using the Gateway LR Clonase® II
system of Invitrogen (FIG. 2), according to the manufacturer's protocol,
yielding the expression construct pcDNA3.1 Hygro TNFR1(ex-tm)-EGFR(cd)
vector.

[0262] Cells expressing the chimeric polypeptides of constructs 4 and 5,
when exposed to the TNF ligand resulted in similar dose response curves
as shown in FIGS. 3a and 3b. Furthermore, similar Z'-value as shown in
FIG. 7 is determined.

[0264] For preparing these constructs and expression vectors, standard
cloning techniques were used according to manufacturer's instructions.

[0265] The resulting PCR product (construct 6) encoding the chimeric
receptor was inserted into the pDONR221 vector of Invitrogen using the
Gateway BP Clonase® enzyme mix (Invitrogen), according to the
manufacturer's protocol.

[0266] To generate the appropriate expression vector the Gateway
cassette® (Invitrogen) was inserted into the ECORV site of the
pcDNA3.1 hygromycin vector (Invitrogen), using standard cloning
techniques. The chimeric receptor DNA was introduced into the expression
vector pcDNA3.1 hygro GW using the LR Clonase® II enzyme mix of
Invitrogen (according to the same principle as schematically illustrated
in FIG. 1b for TNFR1), according to the manufacturer's protocol, yielding
the expression construct pcDNA3.1 hygro DR3(fl)-PDGFR(cd) vector.

Transfection of HEK293T Aequorin Cells and Expression of the Chimeric
DR3(fl)-PDGFR(cd) Receptor

[0267] The HEK293T cells expressing Apoaequorin ("Aequorin cells") were
transfected as described in Examples 1-3 so as to express the chimeric
receptors DR3(fl)-PDGFR(cd) of Example 11.

[0268] In particular, the HEK293T Apoaequorin cells were transfected with
pcDNA3.1 hygro DR3(fl)-PDGFR(cd) vector as prepared in Example 11 using
Optifect® Transfection Reagent (Invitrogen), according to the
manufacturer's protocol.

[0269] Cell surface expression of the chimeric receptors comprising full
length DR3 and the cytoplasmic domain of PDGFR was detected by flow
cytometry. Briefly, cells were harvested and incubated with a PE-labeled
monoclonal antibody directed against DR3 (clone JD3, BD Biosciences) or a
PE-labeled isotype matched mouse IgG (both purchased from BD
Biosciences). Subsequently, cells were washed twice and resuspended in a
final volume of 500 μl. All antibody incubations were performed in
flow cytometry buffer (PBS containing 5% FBS and 0.01% sodium azide) for
20 minutes at 4° C. Flow cytometry was performed using a
FACSCalibur and results were analyzed using Cellquest software (BD
Biosciences, San Jose, Calif.).

[0270] The flow-cytometrical results are shown in FIG. 9, where the black
solid line corresponds to anti-DR3 mAb staining and the dotted line
corresponds to the values obtained with the isotype control.

[0271] These results show that the extracellular domain of DR3 of the
chimeric receptor is found at the surface of the transfected HEK293T
Aequorin cells.

Example 13

Detection of Intracellular Calcium Levels in an HTS Setting of the
Chimeric DR3(fl)-PDGFR(cd) Receptor

[0272] HEK293T cells expressing Apoaequorin and the chimeric
DR3(fl)-PDGFR(cd) receptor (Examples 11 and 12) were plated in 384-well
plates at a concentration of 12500 cells per well in a final volume of 50
μl. The next day culture supernatants were removed and 25 μl
labeling buffer (DMEM:F12 plus 0.1% BSA) containing 2.5 μM
Coelenterazine h (Dalton Pharma services), was added. Cells were
incubated at room temperature for 6 h. A FDSS7000 reader from Hamamatsu
(Japan) was used to examine intracellular calcium levels. This instrument
is designed for high throughput screening and high throughput analysis.
The instrument features include detection with a camera of fluorescence
or luminescence and automatically converts fluorescence or luminescence
signals into numeric data. This digital data is then used to determine
the concentration of calcium inside the analyzed cells. The information
is automatically analyzed in terms of a positive, negative or null effect
of each test compound being examined. This system allowed differences
between untreated and treated cells to be measured, for example by
measuring the calcium flux in cells.

[0273] After a baseline reading of 10 seconds, cells were incubated for 3
minutes with buffer controls. Subsequently cells were stimulated with
TL1A and measurements were continued for another 10 minutes. The results
were analyzed using the FDSS analysis software from Hamamatsu.

[0274] FIG. 10 depicts the dose response curve obtained by exposing the
cells of Example 12 to increasing concentrations of TL1A. FIG. 10 is
established on the basis of the integration of the luminescence emitted
in 10 minutes following administration of TL1A (exposure time) in
dependence of the applied TL1A concentration (AUC). The concentration of
TL1A ranged from 1 ng/ml to 2 μg/ml. The results are representative of
three independent experiments and the error bars represent the standard
deviation of triplicate wells.

[0277] For preparing these constructs and expression vectors, standard
cloning techniques were used according to the same principle as
illustrated in the above examples.

[0278] The HEK293T cells expressing Apoaequorin ("Aequorin cells") were
transfected as described in Examples 1-3 and clones were selected so as
to express the chimeric receptors described below:

[0279] FAS (ex and tm)--PDGFR (cd).

[0280] FAS (ex)--PDGFR (tm and cd).

[0281] FAS (fl)--TNFR1 (DD)--PDGFR (cd)

[0282] FAS (ex and tm)--TNFR1 (cp)--PDGFR (cp).

[0283] TNFR2 (fl)--TNFR1 (DD)--PDGFR (cp).

Example 15

Detection of Intracellular Calcium Levels in an HTS Setting of the
Chimeric TNFRSF Receptors of Example 14

[0284] The clonal HEK293T cells expressing Apoaequorin and the chimeric
receptors as described in Example 15 were plated in 384-well plates at a
concentration of 12500 cells per well in a final volume of 50 μl. The
next day culture supernatants were removed and 25 μl labelling buffer
(DMEM:F12 plus 0.1% BSA) containing 2.5 μM Coelenterazine h (Dalton
Pharma services), was added. Cells were incubated at room temperature for
6 h. A FDSS7000 reader from Hamamatsu (Japan) was used to examine
intracellular calcium levels. After a baseline reading of 10 reads, cells
were incubated for 4 minutes with buffer controls. Subsequently, cells
were stimulated with appropriate agonist ligand FASL (Adipogen), or TNF
(Peprotech) and measurements were continued until the response ended. The
results were analyzed using the FDSS analysis software from Hamamatsu.

[0285] FIGS. 12-16 depict the dose response curve obtained by exposing the
cells of to increasing concentrations of agonist ligands FASL, or TNF.
FIGS. 12-16 are established on the basis of the integration of the
luminescence emitted in 8 to 25 minutes following administration of
agonist ligand (exposure time) in dependence of the applied agonist
ligand concentration (AUC). The concentration of agonist ligand ranged
from 10 pg/ml to 10 μg/ml. The results are representative of several
independent experiments and the error bars represent the standard
deviation of duplicate wells.

[0286] These examples show that various types of chimeric polypeptides as
described in the present specification are suitable for drug screening or
testing in an HTS setting. It is also noted that various combinations of
the different constituent partials sequences yield chimeric receptors
that retain the functions of ligand binding, oligomerization, and in the
case of the RTK portion, tyrosine kinase activity specifically following
ligand binding.